ABSTRACT The overall goal of this research proposal is to determine the molecular and structural determinants for dilated cardiomyopathy (DCM) linked to phospholamban (PLN), a membrane protein involved in Ca2+ transport in the sarcoplasmic reticulum (SR). PLN binds and reversibly inhibits the SR Ca-ATPase (SERCA), regulating heart diastole in response to Ca2+ and ?-adrenergic signaling pathways. ?-adrenergic control of PLN via reversible phosphorylation at Ser16 relieves PLN?s inhibitory effects to restore SERCA?s basal activity, while shifts in cytosolic Ca2+ levels modulate PLN interactions with SERCA. PLN?s position at the crossroads of Ca2+ signaling and ?-adrenergic stimulation makes it a key regulator for cardiac output. Here, we propose to analyze the effects of new lethal mutants of PLN (PLNR9H, PLNR9L, and PLNR25C; AIM1) as well as its post-translational modifications (O-linked N-Glycosylation at Ser16 and S-Acylation at Cys36; AIM2) found in patients diagnosed with DCM and understand how they affect Ca2+ cycling. We will employ an integrated approach including biochemical, molecular biology, and spectroscopic methods (FRET, solution NMR, magic angle spinning, and oriented solid-state NMR) in concert with both in cell and in vivo experiments carried out independently by our collaborators. These experiments will determine the cardiotoxic mechanisms of these new mutants and PTMs to obtain a new, unifying regulatory model for cardiac contractility, bridging the Ca2+ and ?-adrenergic signaling pathways. Finally, in AIM3, we will investigate how to reverse PLN inhibito- ry effects as a mean to augment cardiac contractility under pathological conditions. The scientific premise of the latter AIM is based on our recent discovery that single-stranded oligonucleotides modulate PLN?s regula- tion of SERCA. Together, these studies will pave the way for understanding how mutations and PTMs disrupt Ca2+ and/or ?- adrenergic signaling, and how cardiac function may be rescued by restoring Ca2+ homeostatic balance.